The objective of this study was to evaluate how β-naphthoflavone interacts with lipopolysaccharide (LPS) and polyinosinic acid: polycytidylic acid (poly I: C) induced innate immune parameters as well as phase I and phase II detoxification enzymes in head kidney cells isolated from Atlantic cod. β-naphthoflavone is a pure agonist of aryl hydrocarbon receptor (AhR) while LPS and poly I: C are not. β-naphthoflavone was added to head kidney leukocytes alone or together with LPS or poly I: C and the responses were evaluated in terms of protein and gene expression. The results showed that β-naphthoflavone (25 nM), with and without LPS, significantly induced cytochrome P450 (cyp1c) transcription in cod head kidney cells. β-naphthoflavone (100 nM) in the presence of the virus mimic, poly I: C, also increased cyp1c1transcription. LPS induced cyp1c1, cyclooxygenase 2 (cox2), interleukin 1β (IL-1β), interleukin 6 (IL-6) and interleukin 8 (IL-8) transcription, genes that were not affected by the tested β-naphthoflavone concentrations alone. However, β-naphthoflavone (25 and 50 nM) strengthened LPS induced cox2 and IL-8 transcription. Cod head kidney cells exposed to β-naphthoflavone concentrations ranging from 25 to 100 nM, with and without LPS or poly I: C, expressed AhR protein. LPS or β-naphthoflavone (5–50 nM) significantly induced leukotriene B4 (LTB4) secretion compared to control. In conclusion, this study suggests that β-naphthoflavone could interfere with LPS induced immune cell signaling in cod head kidney cells.